1. Field of the Invention
This invention relates to an autoradiographic gene-screening method.
2. Description of Prior Arts
In molecular biology which has been rapidly developed in recent years, it is essential to obtain genetic information on organisms so as to make the function of the organisms or the mechanism of replication clear. For such purpose, it is required to screen genes having the specific genetic information out of the whole genomes and to obtain a large amount of said selected genes. This process is termed "cloning of genes".
Recently, the cloning of genes has been paid much attention in industry, since it makes it possible to increase the production efficiency of useful products including pharmaceuticals such as interferons, foods such as protein, drought-resistant crops, chemical products, etc.
However, the cloning of genes is difficultly performed in the industry, because the ratio of the objective gene to the whole genomes is very low so that the screening of the specific gene is very difficult. Thus, success or failure of the cloning of genes depends upon whether the screening of genes can be efficiently carried out or not. Further, said screening is carried out as an important means for identifying corresponding genes in the genetic diagnosis of hereditary diseases.
As methods for screening genes, various attempts have been made. As typical methods, there can be mentioned: a method which comprises a process of isolating or purifying the objective gene by a chemical analysis procedure such as column chromatography or electrophoresis utilizing the difference of molecular weights, chemical characteristics, etc. of the gene; a biological method which comprises screening the objective gene by utilizing the difference of manifestation character against drug resistance, enzyme activity. etc.; and a probe method which utilizes the ability to form a hybrid between complementary DNAs or RNAs (that is called "hybridization method").
Among these screening methods, the chemical analysis method is hardly employed in practice, since the amount of gene is very small and further the proportion of the objective gene to whole genomes is very small so that it is very difficult to selectively separate the objective gene. The biological method has a disadvantage in that manifestation of function related to the objective gene is indispensable to the method and not always active. As compared with these two methods, the probe method has advantages in that hybridization occurs very selectively and that the procedure is very simple.
For example, the typical gene-screening method utilizing colony hybridization process is carried out in the following manner.
In the first place, a recombinant DNA including a DNA fragment having the objective gene is prepared. In more detail, a plasmid obtained from Escherichia coli which is to serve as a vector is cleaved by a specific restriction enzyme. A DNA or DNA fragment containing the objective gene is also cleaved by the same restriction enzyme so that both the plasmid vector and the DNA have the same cohesive end. Both are then joined to each other at the corresponding ends using DNA ligase, whereby the recombinant plasmid is obtained.
The DNA fragment containing the objective gene can be also obtained, for example, by randomly cleaving a DNA containing the objective gene by a shotgun method and appropriately restoring the end of each of the resulting DNA fragments using DNA polymerase.
In the second place, E. coli is infected with the so-obtained recombinant plasmid and planted on an agar plate culture medium in a dispersed state to form colonies (clones having the same gene produced by proliferation of E. coli ). The agar plate culture medium on which colonies are formed, is hereinafter referred to as master plate.
In the third place, a part of the colonies is transferred onto a fresh filter, for example, by a replica plating method, etc. The resulting filter is hereinafter referred to as replica filter. This replica filter is placed on a fresh agar plate culture medium and incubated until the colonies are grown to an appropriate size. The filter is then peeled off from the agar plate medium and hybridization is carried out thereon.
The hybridization is carried out in the following manner. The cells of E. coli on the filter are lysed and the resulting exposed plasmid DNA is denatured to form a single-stranded DNA which is then fixed on the filter. Separately, DNA or RNA which is complementary to DNA containing the objective gene is radioactively labeled to prepare a probe.
In the fourth place, the radioactively labeled DNA or RNA is then hybridized with the denatured DNA on the filter. Thus, only a hybrid of a DNA containing the objective gene with the radioactively labeled DNA or RNA is formed on the filter and it is at the same time radioactively labeled. The hybridized filter is then subjected to autoradiography, whereby the colonies containing the objective gene are identified and the so identified colonies are collected from the master plate which has been stored at a low temperature.
Accordingly, through the above-described gene-screening procedure (hybridization utilizing) of recombinant genes, the objective gene clones are obtained.
As other methods for screening genes utilizing hybridization, there can be mentioned a screening method utilizing plaque hybridization.
In the plaque hybridization method, phage (virus which invades bacteria through infection and lyses their cells, resulting in growth of the virus) is used as a vector, and a replica of plaque formed on the master plate containing bacteria serving as a host is formed on a filter. Subsequently, a hybrid with a radioactively labeled probe is formed on the filter in a similar manner to that described above.
The above-summerized gene-screening methods utilizing colony hybridization or plaque hybridization are described in more detail in the following literatures.
METHODS IN ENZYMOLOGY, Vol. 68, pp. 379-395 (edited by Ray Wu, ACADEMIC PRESS, NEW YORK, 1979)
PROTEIN, NUCLEIC ACID & ENZYME (in Japanese), Vol. 26, No. 4, pp. 575-583 (1981).
In the conventional autoradiography employed for the gene-screening, a radiographic film such as a high sensitivity radiographic film is combined in layers with a filter retaining a captured radioactively labeled probe for a given time so that the film is exposed to the radiation. A radiographic intensifying screen is generally employed to enhance the detection sensitivity of autoradiography. Such autoradiography is described, for example, in the following literature: Method in Biochemical Experiment, Vol. 6, Method in Tracer Experiment I, pp. 271-280, "B. Autoradiography" by Toru Sueyoshi & Akiyo Shigematsu (Tokyo Kagaku Dozin Ltd., 1977).
Therefore, the autoradiography is an important means for identifying the objective gene and obtaining two dimensional information on the location of said gene in the gene-screening method. Further, it can be said that the autoradiography is a very useful means, since the isolation and purification of the objective gene can be done according to the obtained locational information. Nevertheless, such useful autoradiography is not free from several drawbacks in the practical use when applied to the gene-screening method utilizing the hybridization mentioned above.
As described above, in the conventional autoradiography, a filter containing a radioactively labeled substance is brought into contact in layers with a radiographic film such as a high-sensitivity radiographic film for a given time so that the film is exposed to the radiation and then a visible image indicating the position of the radioactive substances is obtained.
The primary drawback resides in that the exposure operation requires a long period of time. The exposure operation in the conventional autoradiographic screening is usually carried out for several days, and requires at least several tens of hours even when a radiographic intensifying screen is used. This is because the amount of DNA fixed on the filter is small and the radioactively labeled substance (radioactively labeled probe) is a DNA and RNA partially labeled generally with .sup.32 P so that intense radioactivity is not imparted thereto.
The second drawback resides in that the exposure operation should be carried out usually at a low temperature, for example, a temperature in the range of 0.degree. C. to -0.degree. C. This is because a latent image on silver salt which is formed by exposure to a radiation or fluorescence on the film tends to fade at a relatively high temperature such as room temperature and the so degraded latent image can be no longer developed to give a readable image. Further, the silver salt is easily fogged chemically through migration of deleterious ingredients from the hybridization-processed filter to the silver salt layer at such a high temperature. Another reason resides in that the silver salt difficultly forms a latent image at a relatively high temperature such as room temperature in the case of utilizing an intensifying screen which gives an emission of low intensity.
The third drawback resides in that the exposure ought to be carried out in a dry state to prevent the radiographic film from wetting and being fogged. Usually, this is done after the filter is dried, othewise enclosed with a synthetic resin wrapping film, etc.
When the image obtained by the autoradiography is fogged as described above, the hybridized DNA is hardly distinguished on the obtained image and hence, the result of screening is made remarkably unfavorable.
For these reasons, the operation involved in the conventional autoradiography is complicated, whereby the gene-screening procedure is made complicated as a whole.
Other drawbacks of the conventional autoradiographic gene-screening method are given below.
The photosensitive silver salt of the radiographic film is readily influenced by physical irritation and the radiographic film easily produces fogging under application of physical pressure caused by the contact of the film with the hands of operators or the instrument in the exposure operation. Such unfavorable phenomena also cause lowering in accuracy of the gene-screening. In order to avoid the occurrence of physical fogging of the radiographic film, much skill and caution must be taken in the handling of the film, and hence, the screening operation is further complicated.
The exposure operation in the conventional autoradiographic gene-screening method is conducted over a long period of time as described above, so that it is unavoidable that the radiations of natural origin and radioactive impurities incorporated in the filter in addition to the radioactively labeled substance take part in the exposure of the radiographic film. Thus, the accuracy of the resulting information on the location of the objective radioactively labeled substance is lowered. In order to eliminate such interference and to set appropriate exposure conditions, parallel experiments using control samples are generally carried out to find out exposure time, but such experiments have disadvantages in that the number of the experiments is increased because of involving such parallel experiments and preliminary experiments for ascertaining appropriate exposure time and hence, the operation is made complicated and less economical as a whole.
The operation of collecting the objective gene is performed in such a manner that the master plate is alligned with the autoradiograph to allow the clone (colonies and plaques) corresponding to positive-signals indicating the presence of the radioactively labeled substances to be picked up. Therefore, if the formation of clones on the culture medium is insufficient, the hybridization is insufficient, or the condition for preparing the replica filter or for exposure of the filter is improper, the accuracy of the gene-screening is lowered. Otherwise, the screening becomes impossible in some cases and accordingly the number of the screening operation involved necessarily increases.